Faced with a pathogen too dangerous to control using traditional pathogen-based vaccines, we have developed an alternative, safe, live-virus vaccine system. Our laboratory has been producing combinatorial libraries of human rhinoviruses (HRVs) that display HIV-1 immunogens connected to their surface via linkers of varied lengths and sequences. The resulting diversity of presentations increases the chances of generating useful immunogens. Anti-HIV-1 neutralizing antibodies can be exploited to select for viruses that present their HIV epitopes in ways most like HIV. Using this approach, we have been able to generate viruses that display the HIV-1 gp41 ELDKWA epitope (that corresponds to the broadly neutralizing anti-HIV-1 antibody, 2F5) in ways that elicit potent and cross-reactive neutralizing responses against diverse isolates of HIV-1 (from clades A, B, D, and E). This result represents a significant advance toward AIDS vaccine development, as these viruses are the first recombinant HIV-based immunogens to elicit broadly neutralizing antibodies. With the goal of developing the most effective immunogens possible, we propose to: (1) generate and select for improved anti-HIV-1 immunogens from combinatorial libraries displaying membrane-proximal external region (MPER) segments corresponding to: (i) the ELDKWA epitope, designed in response to what was learned from the ELDKWA library that yielded valuable immunogens, (ii) the NWFDITNW epitope (adjacent to the ELDKWA epitope) that corresponds to the most broadly neutralizing anti-HIV-1 antibody known, 4E10, and (iii) both the ELDKWA and NWFDITNW epitopes (in their native, more extended MPER sequence);(2) generate and select for new neutralizing mAbs from rabbits and mice immunized with the most broadly immunogenic MPER-presenting viruses ([unreadable] peptide boosts), and (3) determine the three-dimensional structures of our most promising engineered viruses and/or peptide boosts, free and complexed with neutralizing anti-HIV-1 Fab fragments. For this, we will use crystallography, cryoelectron microscopy, and NMR, not knowing in advance which techniques will be most fruitful for characterizing the structures and dynamics of the immunogens. We have learned from determining the structures of a number of immunogenic V3 loop-presenting viruses that the epitopes displayed are often in more than one conformation, demanding broad technical approaches and thinking to go with it. By learning more about the three-dimensional determinants of antigenicity and immunogenicity, we hope to be able to approach structure-based vaccine design in a way that eventually will parallel the successful paradigm of structure-based drug design.